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*12-O-TETRADECANOYLPHORBOL-13-ACETATE
The Journal of Immunology, 2001, 167: 3164-3173.
Copyright © 2001 by The American Association of Immunologists

Disruption of NF-{kappa}B Signaling Reveals a Novel Role for NF-{kappa}B in the Regulation of TNF-Related Apoptosis-Inducing Ligand Expression1

Tudor M. Baetu*,{dagger},{ddagger}, Hakju Kwon*,{dagger}, Sonia Sharma*,{dagger},{ddagger}, Nathalie Grandvaux*,{dagger} and John Hiscott2,*,{dagger},{ddagger},§

* Terry Fox Molecular Oncology Group, {dagger} Lady Davis Institute for Medical Research, and Departments of {ddagger} Microbiology and Immunology and § Medicine and Oncology, McGill University, Montreal, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The NF-{kappa}B family of transcription factors functions broadly in the host control of immunoregulatory gene expression, inflammation, and apoptosis. Using Jurkat T cells engineered to inducibly express a transdominant repressor of I{kappa}B{alpha}, we examined the role of NF-{kappa}B in the regulation of cytokine and apoptotic gene expression. In this T cell model, as well as in primary T lymphocytes, expression of TNF-related apoptosis-inducing ligand (TRAIL) apoptotic signaling protein was dramatically down-regulated by inhibition of NF-{kappa}B binding activity. TRAIL acts through membrane death receptors to induce apoptosis of activated T lymphocytes and can be up-regulated by a variety of physiological and pharmacological inducers. However, regulation of TRAIL gene expression has not been defined. Treatment with TCR mimetics (PMA/ionomycin, PHA, and anti-CD3/CD28 Abs) resulted in a rapid increase in the expression of TRAIL mRNA and cell surface TRAIL protein. Induction of the transdominant repressor of I{kappa}B{alpha} dramatically down-regulated surface expression of TRAIL, indicating an essential role for NF-{kappa}B in the regulation of TRAIL. The induced expression of TRAIL was linked to a c-Rel binding site in the proximal TRAIL promoter at position -256 to -265; mutation of this site or an adjacent {kappa}B site resulted in a complete loss of the inducibility of the TRAIL promoter. The regulation of TRAIL expression by NF-{kappa}B may represent a general mechanism that contributes to the control of TRAIL-mediated apoptosis in T lymphocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL),3 also known as Apo2 ligand, is a 40-kDa type II transmembrane protein that is structurally related to the TNF family of proteins. The extracellular domain of TRAIL shares the highest amino acid homology with the Fas ligand (28%), TNF-{alpha} (23%), lymphotoxin-{alpha} (23%), and lymphotoxin-{beta} (22%) (1, 2). To date, several TRAIL receptors have been identified. Two of these receptors TRAIL-R1/death receptor 4 and TRAIL-R2/death receptor 5 contain cytoplasmic death domains and signal apoptosis through a caspase-dependent pathway (3, 4, 5, 6). Engagement of either of these two receptors by TRAIL results in the recruitment and activation of caspase-8, as well as cleavage of BH3-interacting death domain agonist and cytochrome c release from mitochondria, events that subsequently lead to the activation of the caspase cascade (7, 8). As yet, it is not clear whether TRAIL-R1 and TRAIL-R2 signal apoptosis through the Fas-associated death domain (FADD) protein. According to some studies, a dominant mutant of FADD abolishes TRAIL-mediated apoptosis (9, 10, 11); however, fibroblasts derived form FADD-deficient mice undergo apoptosis upon overexpression of TRAIL-R1, suggesting the existence of a FADD-independent signaling mechanism (12). Binding of TRAIL to TRAIL-R1 or TRAIL-R2 also results in the activation of NF-{kappa}B, indicating that TRAIL receptors can signal both apoptosis and gene transcription; however, activation of NF-{kappa}B alone is not sufficient to block apoptosis induced by TRAIL receptors (9, 11). More recent studies suggest that TRAIL-induced activation of NF-{kappa}B is mediated by a TNFR-associated factor 2/NF-{kappa}B-inducing kinase (NIK)/I{kappa}B kinase (IKK){alpha} and {beta}-dependent signaling cascade (13). In contrast with TRAIL-R1 and TRAIL-R2, TRAIL-R3/decoy receptor (DcR)1 exists as a GPI-anchored surface protein that is unable to signal cell death, thus acting as a decoy receptor (4, 7, 14, 15, 16). A fourth TRAIL receptor, TRAIL-R4/DcR2 contains only a partial death domain and does not mediate apoptosis upon binding of TRAIL; this member retains the ability to activate NF-{kappa}B, suggesting that it may inhibit TRAIL-induced apoptosis by inducing antiapoptotic genes (17). Transfection of nonsignaling TRAIL-R3 or TRAIL-R4 results in a down-regulation in the amount of cell death. Furthermore, TRAIL-R3 mRNA is preferentially found in normal cells but not in transformed cells, suggesting that these DcRs might be responsible for the resistance of normal cells to TRAIL-induced apoptosis (4, 5, 14). These results suggest a complex regulation of TRAIL-induced apoptosis at the level of expression of the various TRAIL receptors.

Although TRAIL mRNA is detected in various cells and tissues, including PBLs, spleen, and thymus (1, 2), regulation of its expression remains largely unknown. Previous experiments have shown that type I IFNs, as well as IL-2 and IL-15 stimulation, induced expression of TRAIL by NK cells (18, 19), and that the constitutive expression of TRAIL on liver NK cells may be dependent on the endogenous production of IFN-{gamma} (20). In addition, T cells activated via the TCR or PMA/ionomycin or PHA stimulation display increased levels of TRAIL mRNA (21, 22, 23). It has also been reported that peripheral blood T cells stimulated with anti-CD3 Ab and type I IFNs display an increase in TRAIL expression (19).

The NF-{kappa}B/Rel family of transcription factors plays an essential role in the regulation of a number of genes involved in pathogen response, immunomodulation, cell growth regulation, and apoptosis. NF-{kappa}B activity is controlled by the inhibitory I{kappa}B proteins, which mask the nuclear localization sequence in the Rel homology domain of NF-{kappa}B, thereby sequestering NF-{kappa}B in a latent state in the cytoplasm (24, 25, 26). The best characterized I{kappa}B protein is I{kappa}B{alpha}, which is composed of three domains: an N-terminal signal-responsive domain, a central ankyrin repeat domain that interacts with NF-{kappa}B, and a C-terminal PEST domain that is responsible for the basal turnover of the protein (27, 28, 29, 30, 31, 32). Upon stimulation with agents such as TNF-{alpha} and PMA, I{kappa}B{alpha} is phosphorylated at Ser32 and Ser36 in the N-terminal signal responsive domain by the IKK complex (33, 34, 35). Phosphorylated I{kappa}B{alpha} is subsequently polyubiquitinated by pI{kappa}B{alpha}-ubiquitin E3 ligase (36, 37) and targeted to the 26S proteasome complex, resulting in the release and nuclear translocation of NF-{kappa}B, which can now stimulate target gene transcription. Mutation of the I{kappa}B serine phosphorylation sites to alanines (Ser32,36 Ala) generates a form of I{kappa}B that is no longer responsive to inducer-meditated phosphorylation and degradation and thus acts as a transdominant repressor of the NF-{kappa}B pathway (38, 39, 40, 41, 42, 43). In this study, we characterized the expression of TRAIL in Jurkat T cells both at the mRNA and protein levels. Using Jurkat cells inducibly expressing I{kappa}B{alpha} Ser32,36 Ala (a transdominant repressor of I{kappa}B{alpha}, TD-I{kappa}B{alpha}) (44, 45), we provide evidence that the induced expression of TRAIL on T cells is dependent on NF-{kappa}B.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and reagents

The reverse tetracycline transactivator protein (rtTA)-Neo and rtTA-I{kappa}B{alpha}(2N{Delta}4) Jurkat T cells were described previously (44). Primary T cells were extracted from human whole blood using the RosetteSep CD3+ cell enrichment kit (StemCell Technologies, Vancouver, Canada) according to the manufacturer’s instructions. The estimated cell purity of the CD3+ lymphocyte population obtained is 94% or more. All cells were grown in RPMI 1640 containing 10% FBS, 2 mM glutamine, and 10 µg/ml gentamicin. Cells were stimulated by TNF-{alpha} (20 µg/ml; R&D Systems, Minneapolis, MN) or PMA (100 ng/ml; ICN Pharmaceuticals, Costa Mesa, CA) alone or in combination with PHA (1 µg/ml), Con A (10 µg/ml), or ionomycin (200, 500, or 1000 pmol/ml). CD3/CD28 stimulation of Jurkat T cells was achieved by treating 106 cells with a mixture of anti-CD3 (10 ng/ml) and anti-CD28 (5 µg/ml) Abs (BD PharMingen, San Diego, CA) for 48 h. De novo protein synthesis was blocked by treating cells with cyclohexamide at various concentrations (25, 50, and 100 µg/ml). Primary T lymphocytes were exposed to various NF-{kappa}B inhibitors (Calbiochem, San Diego, CA) as follows: sodium salicylate (NaSal, 20 mM for 24 and 48 h), Bay 11-7082 (5 µM for 24 h), and MG132 (10 µM for 4 h).

RNase protection assay

Ten micrograms of total RNA extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA) from unstimulated or stimulated rtTA-Neo or rtTA-I{kappa}B{alpha}(2N{Delta}4) Jurkat T cells was subjected to RNase protection assay as specified by the manufacturer using APO3 and hAPO3c probe sets (BD PharMingen). The resulting protected RNAs were resolved by 5% denaturing gel and exposed to x-ray film.

Immunoblot analysis

To characterize the kinetics of expression, rtTA-Neo and rtTA-I{kappa}B{alpha}(2N{Delta}4) Jurkat T cells were cultured in in the presence of 1 µg/ml doxycycline (Dox; Sigma-Aldrich, St. Louis, MO) for various amounts of time. Cells were then washed with PBS and lysed in the presence of 1 µg/ml KCl, 1 mM EDTA, 1 mM DTT, 0.5% Nonidet P-40 (NP-40), 0.5 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 10 µg/ml aprotinin. Equivalent amounts of whole-cell extract were subjected to SDS-PAGE in a 10% polyacrylamide gel. After electrophoresis, the proteins were transferred to Hybond transfer membrane (Amersham, Arlington Heights, IL) in a buffer contaning 30 mM Tris, 200 mM glycine, and 20% methanol for 1 h. The membrane was blocked by incubation in PBS containing 5% dried milk for 1 h and then incubated overnight at 4°C with N-terminal I{kappa}B{alpha} mAb MAD 10B (30) and TRAIL-specific mAb (Immunex, Seattle, WA) in 5% milk-PBS at dilutions of 1/1000. After four 10-min washes with PBS, membranes were incubated with a peroxidase-conjugated secondary goat anti-mouse Ab (Amersham) at a dilution of 1/1000. The reaction was then visualized with the ECL system as recommended by the manufacturer (Amersham).

The EMSA

Following the addition of 1 µg/ml Dox to the culture medium for 24 h, nuclear extracts were prepared from rtTA-Neo and rtTA-I{kappa}B{alpha}(2N{Delta}4) Jurkat T cells after induction with TNF-{alpha} (10 ng/ml) and PMA (50 ng/ml) for 0–24 h. Cells were washed in buffer A (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.5 mM PMSF) and were resuspended in buffer A containing 0.1% NP-40. Cells were then chilled on ice for 10 min before centrifugation at 10,000 x g. Pellets were then resuspended in buffer B (20 mM HEPES (pH 7.9), 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml pepstatin, 0.5 mM spermidine, 0.15 mM spermine, and 5 µg/ml aprotinin). Samples were incubated on ice for 15 min before centrifugation at 10,000 x g. Nuclear extract supernatants were diluted with buffer C (20 mM HEPES (pH 7.9), 20% glycerol, 0.2 mM EDTA, 50 mM KCl, 0.5 mM DTT, and 0.5 mM PMSF). Nuclear extracts were subjected to EMSA with a 32P-labeled probe corresponding to the {kappa}B1 region of the I{kappa}B{alpha} promoter (5'-GATCTTGGAAATTCCCCGA-3') or the {kappa}B1 (5'-AAAGCAAAGAAAATCCCTCCCCT-3') and the mutant {kappa}B1 (5'-AAAGCAAAGTCAAAACCTCCCCT-3') sites of the TRAIL promoter. The NF-{kappa}B binding sites are underlined. Supershift analysis was performed using the c-Rel, p50, p65, and RelB Abs (Santa Cruz Biotechnology, Santa Cruz, CA). The resulting protein-DNA complexes were resolved by 5% Tris-glycine gel and exposed to x-ray film.

Immunoprecipitation

The rtTA-Neo and rtTA-I{kappa}B{alpha}(2N{Delta}4) Jurkat cells were induced with TNF-{alpha} (10 ng/ml) and PMA (50 ng/ml) for various times in the presence or absence of Dox and were lysed in TNN buffer (20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 0.5% NP-40, 2 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml pepstatin, 0.5 mM spermidine, 0.15 mM spermine, and 5 µg/ml aprotinin). Cell lysates (500 µg) were precleared with preimmune sera. Precleared lysates were incubated with 10 µl p65 Ab or I{kappa}B{alpha} Ab and 30 µl protein A-Sepharose beads (Amersham Pharmacia Biotech, Upssala, Sweden) for 1 h at 4°C. Beads were washed five times with TNN buffer, and the immunoprecipitates were eluted by boiling the beads for 5 min in SDS loading dye. Eluted proteins were electrophoresed on 10% SDS-PAGE and were detected by using anti-goat p65 Ab (Santa Cruz Biotechnology) and MAD10B I{kappa}B{alpha} Ab.

Flow cytometric analysis

Unstimulated and stimulated rtTA-Neo and rtTA-I{kappa}B{alpha}(2N{Delta}4) Jurkat T cells (2 x 106 cells) were washed in cold 0.5% BSA in PBS and resuspended in PBS containing 10% goat serum and 0.5% BSA for 15 min. Cells were pelleted and incubated with 1 µg monoclonal anti-TRAIL Ab (Immunex) for 1 h at room temperature and were then washed with 0.5% BSA in PBS and incubated for 45 min with FITC-conjugated anti-mouse secondary Ab (1/100; Sigma-Aldrich). After washing with PBS, cells were subjected to flow cytometric analysis. Data were collected (5000 events) using a Coulter EPICS XL-MCL (Beckman Coulter, Fullerton, CA) and analyzed with CellQuest (BD Biosciences, Mountain View, CA) and WinMDI software (version 2.8, copyright Joseph Trotter).

Promoter cloning and mutations

The TRAIL promoter region was cloned from human genomic DNA isolated using the Qiagen DNeasy tissue kit according to the manufacturer’s instructions. The full length 1578-bp TRAIL promoter fragment was PCR cloned using the 5'-GATCCTGTCAGAGTCTGACTGCTGTAAGT-3' sense and 5'-GTAGACTCATTTACAGATAGAAGGCAAGG-5' antisense primers. The -483- and -1002-bp regions were cloned using the 5'-AGCAAGACCATTGCTATG-3' and 5'-CTCCAGCCTGGGCGATAAA-3' sense primers together with the 5'-GTAGACTCATTTACAGATAGAAGGCAAGG-3' antisense primer. The {kappa}B1 and {kappa}B2 mutants of the TRAIL promoter were generated by overlap PCR. The {kappa}B1 sequence was mutated from 5'-AGAAAATCCC-3' to 5'-AGTCAAAACC-3', and the {kappa}B2 sequence from 5'-TGGAAGTTTC-3' to 5'-TGTCAGAATC-3'. All constructs were cloned into the NheI/KpnI site of the basic pGL3 luciferase vector and into the XbaI/PstI site of the basic pCAT vector, and the sequences were confirmed by DNA sequencing.

Transient transfection and reporter assays

Jurkat T cells (106 cells/transfection) were cultured at 5 x 105 cells/ml the day before transfection. TRAIL promoter constructs (1 µg) were transfected with 100 ng pRL-tk using the FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) as indicated by the manufacturer. After 48 h, luciferase activity was measured using a dual luciferase reporter assay system (Promega, Madison, WI). For chloramphenicol acetyltransferase (CAT) assays, 107 cells/transfection were transfected by electroporation with 10 µg reporter construct and 1 µg pRL-tk. Following induction, CAT activity was measured. pRL-tk was used to normalize the transfection efficiencies.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TD-I{kappa}B{alpha} inhibits NF-{kappa}B binding to {kappa}B1 site of the I{kappa}B{alpha} promoter

To specifically block NF-{kappa}B activation, a T cell line that inducibly expresses TD-I{kappa}B{alpha} was generated (44, 45). For this purpose, Jurkat T cells selected for the expression of the rtTA (46) were stably transfected with a CMVt-Neo construct expressing the 2N{Delta}4 I{kappa}B{alpha} mutant in which both Ser32 and Ser36 were mutated to alanines. Such an I{kappa}B protein cannot be phosphorylated and targeted to degradation, and the NF-{kappa}B dimers are kept inactive within the cytoplasm following the activation of the IKK complex. In addition, this I{kappa}B{alpha} mutant also contains a small 22-aa C-terminal deletion, which was generated to distinguish the mutated I{kappa}B{alpha} from the endogenous I{kappa}B{alpha} protein; this deletion is dispensable with regard to NF-{kappa}B binding and function (44). A control cell line (rtTA-Neo) containing the empty CMVt-Neo vector was also generated.

Initially, the effect of TD-I{kappa}B{alpha} on TNF-{alpha} expression and PMA-induced NF-{kappa}B binding activity was studied by EMSA using the {kappa}B1 site of the I{kappa}B{alpha} promoter as a probe (44, 45). Treatment of rtTA-Neo Jurkat cells with TNF-{alpha} or PMA/PHA resulted in a strong induction of NF-{kappa}B binding activity, irrespective of Dox treatment (Fig. 1Go. A and B, lanes 1–4 and 6–9). In contrast, TNF-{alpha}- or PMA/PHA-induced NF-{kappa}B binding activity in rtTA-I{kappa}B{alpha}(2N{Delta}4) Jurkat cells was completely blocked by Dox induction of TD-I{kappa}B{alpha} (Fig. 1Go, A and B, lanes 6–9), whereas NF-{kappa}B binding activity was observed in rtTA-I{kappa}B{alpha}(2N{Delta}4) Jurkat cells without Dox treatment (Fig. 1Go, A and B, lanes 1–4). To identify the subunit composition of the NF-{kappa}B complexes, supershift analysis of extracts from rtTA-Neo Jurkat cells treated with TNF-{alpha} and PMA for 4 h was performed using anti-p65, -p50, -p52, and -c-Rel Abs. Complexes were shifted mainly with p65 or p50 Abs but not with p52 or c-Rel Abs (data not shown). Thus, NF-{kappa}B DNA binding activity was completely blocked by expressing TD-I{kappa}B{alpha}.



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  		FIGURE 1. TD-I{kappa}B{alpha} blocks NF-{kappa}B DNA binding activity. A, Nuclear extracts from rtTA-Neo (left panel) and rtTA-I{kappa}B{alpha}(2N{Delta}4) (right panel) Jurkat cells treated with TNF-{alpha} (10 ng/ml) for the indicated times in the absence (lanes 1–4, 10–14) or presence (lanes 6–9, 15–18) of Dox pretreatment (1 µg/ml, 24 h) were subjected to an EMSA using the {kappa}B1 probe as described previously (44 ). B, Nuclear extracts from rtTA-Neo (left panel) and rtTA-I{kappa}B{alpha}(2N{Delta}4) (right panel) Jurkat cells treated with PMA (50 ng/ml)/PHA (1 µg/ml) for the indicated times in the absence (lanes 1–4, 10–14) or presence (lanes 6–9, 15–18) of Dox pretreatment (1 µg/ml, 24 h) were subjected to an EMSA using the {kappa}B1 probe. Control (C) lanes (lanes 5 and 14 in A and B) represent the competition of NF-{kappa}B-DNA complex formation using a 125-fold excess of unlabeled {kappa}B1 probe.

 
p65 is tightly bound to TD-I{kappa}B{alpha}

Coimmunoprecipitation studies were performed with anti-p65 (Fig. 2Go, A and B) and anti-I{kappa}B{alpha} Abs (Fig. 2GoC) to determine whether TD-I{kappa}B{alpha} could associate with p65 in vivo during the course of TNF-{alpha} or PMA/PHA induction. In unstimulated rtTA-Neo Jurkat cells, p65 complexed with I{kappa}B{alpha} in the presence or absence of Dox (Fig. 2Go, A and B, lanes 1 and 5). In unstimulated rtTA-I{kappa}B{alpha}(2N{Delta}4) Jurkat cells without Dox pretreatment, p65 was associated with both endogenous I{kappa}B{alpha} and TD-I{kappa}B{alpha} (Fig. 2Go, A and B, lane 10), whereas in Dox-treated TD-I{kappa}B{alpha} expressing cells, p65 was mainly associated with TD-I{kappa}B{alpha} (Fig. 2Go, A and B, lane 14), due to down-regulation of endogenous I{kappa}B{alpha} expression (44, 45). TNF-{alpha} or PMA/PHA stimulation induced degradation of I{kappa}B{alpha}, which reappeared after 60 min in control Jurkat cells (Fig. 2Go, A and B, lanes 2–4 and 6–8) in the presence or absence of Dox; for rtTA-I{kappa}B{alpha}(2N{Delta}4) Jurkat cells in the absence of Dox, the same loss and reappearance of endogenous I{kappa}B{alpha} was observed (Fig. 2Go, A and B, lanes 11–13). In contrast, in TD-I{kappa}B{alpha}-expressing Jurkat cells, immunoprecipitation with anti-p65 Ab resulted in the coimmunoprecipitation of predominantly I{kappa}B{alpha} 2N{Delta}4 (Fig. 2Go, A and B, lanes 14–17), indicating that the degradation-resistant TD-I{kappa}B{alpha} was tightly associated with p65 throughout induction. The reciprocal immunoprecipitation with anti-I{kappa}B{alpha} Ab was also performed (Fig. 2GoC). Both endogenous and TD-I{kappa}B{alpha} were immunoprecipitated from rtTA-I{kappa}B{alpha}(2N{Delta}4)-expressing cells in the absence of Dox addition, together with p65 (Fig. 2GoC, lanes 2–5), whereas following Dox treatment, only TD-I{kappa}B{alpha} and associated p65 were identified (Fig. 2GoC, lanes 7–9). Therefore, inhibition of NF-{kappa}B DNA-binding activity in TD-I{kappa}B{alpha}-inducible cells is due to the tight association between the NF-{kappa}B transactivator p65 and TD-I{kappa}B{alpha}, which is resistant to inducer-mediated degradation.



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  		FIGURE 2. TD-I{kappa}B{alpha} is tightly bound to RelA (p65). The rtTA-Neo (lanes 1–8) and rtTA-I{kappa}B{alpha}(2N{Delta}4) (lanes 9–17) Jurkat cells were treated with TNF-{alpha} (10 ng/ml) (A) or PMA (50 ng/ml)/PHA (1 µg/ml) (B) for various times in the absence (lanes 1–4 and 9–13) or presence (lanes 5–8 and 14–17) of Dox (1 µg/ml) for 24 h. Cell lysates were immunoprecipitated with p65-specific Ab, and immunoblot analysis was performed using the anti-I{kappa}B{alpha} and anti-p65 Abs. Arrows indicate p65, I{kappa}B{alpha}, and I{kappa}B{alpha}(2N{Delta}4) from top to bottom. Control (P) lane (lane 9) represents an immunoprecipitation with rabbit preimmune sera. C, The rtTA-I{kappa}B{alpha}(2N{Delta}4) Jurkat cells were treated with PMA (50 ng/ml)/PHA (1 µg/ml) for various times in the absence (lanes 1–5) or presence (lanes 6–9) of Dox (1 µg/ml, 24 h). Cell lysates were immunoprecipitated with I{kappa}B{alpha}-specific Ab. Western blot analysis was done using the anti-p65 and anti-I{kappa}B{alpha} Abs. Arrows indicate p65, I{kappa}B{alpha}, and I{kappa}B{alpha}(2N{Delta}4) from top to bottom. Control (P) lane (lane 1) represents an immunoprecipitation with mouse preimmune sera.

 
Suppression of apoptosis-related gene expression by TD-I{kappa}B{alpha}

To identify novel NF-{kappa}B-regulated cytokine and apoptosis-related genes, rtTA-Neo and rtTA-I{kappa}B{alpha}(2N{Delta}4) Jurkat T cells were incubated in the absence or presence of Dox for different times and were subsequently treated with TNF-{alpha} or PMA for 4 h. RNase protection assays using RNA extracted from these cells revealed that the amounts of TRAIL mRNA increased upon treatment with both TNF-{alpha} (Fig. 3GoA, lanes 3–7 and 15) and PMA (Fig. 3GoA, lanes 8–12 and 20). As a control for the rtTA-Neo Jurkat cells, untransfected Jurkat cells were also tested for TRAIL expression; in both cell lines, TNF-{alpha} and PMA stimulated TRAIL mRNA levels (data not shown). As displayed in Fig. 3GoA, the increased expression of TRAIL mRNA observed upon induction with PMA or TNF-{alpha} was almost completely suppressed by inducing TD-I{kappa}B{alpha} expression in rtTA-I{kappa}B{alpha}(2N{Delta}4) cells (Fig. 3GoA, compare lane 15 with lanes 16–19 and lane 20 with lanes 21–24). In both cases, Dox treatment alone did not affect gene expression patterns (Fig. 3GoA, lanes 1, 2, 13, and 14). As little as 6 h of Dox treatment before TNF-{alpha} or PMA stimulation was sufficient to abrogate TRAIL mRNA expression. By comparison, Fas, whose expression is known to be dependent on NF-{kappa}B, was also up-regulated upon TNF-{alpha} and PMA stimulation and down-regulated once TD-I{kappa}B{alpha} was expressed (Fig. 3GoA, lanes 15–24). A modest inhibition of receptor-interacting protein was also noted. To correlate the decrease in TRAIL expression with the increase in TD-I{kappa}B{alpha} expression, protein extracts from the same cells were subjected to immunoblot analysis; as shown in Fig. 3GoB, increased expression of TD-I{kappa}B{alpha} correlated with a decrease in endogenous I{kappa}B{alpha} (44) (Fig. 3GoB, compare lanes 6 and 7), and Dox treatment before PMA stimulation correlated with the reduction in TRAIL mRNA (Fig. 3Go, A, lanes 20–24, and B, lanes 8–10). These initial observations suggest a possible role for NF-{kappa}B as a regulator of TRAIL in lymphocytes.



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  		FIGURE 3. Suppression of apoptosis-related gene expression by TD-I{kappa}B{alpha}. A, The rtTA-Neo and rtTA-I{kappa}B{alpha}(2N{Delta}4) Jurkat cells were incubated with Dox for different intervals (0–48 h) and subsequently treated with TNF-{alpha} (20 ng/ml; lanes 3–7 and 15–19) and PMA (100 ng/ml; lanes 8–12 and 20–24) for 4 h. Total RNA was extracted and then subjected to RNase protection assay using the hAPO3c probe set (RiboQuant, BD PharMingen). M, Unprotected probes. L32- and GAPDH-protected probes were used for normalization. B, The rtTA-Neo and rtTA-I{kappa}B{alpha}(2N{Delta}4) Jurkat cells were incubated with Dox for different intervals (0–24 h) and subsequently treated with PMA (100 ng/ml; lanes 3–5 and 8–10) for 4 h. Expression of I{kappa}B{alpha} was analyzed by immunoblotting using the I{kappa}B{alpha} MAD10B-specific Ab.

 
Expression of TRAIL protein on Jurkat T cells

Next we sought to correlate TRAIL mRNA expression levels with the amount of TRAIL protein expressed by stimulated cells. TRAIL protein expression was detected both in whole-cell extracts by immunoblot analysis as well as at the level of cell surface expression by flow cytometric analysis. PMA stimulation of Jurkat T cells resulted in a significant increase in TRAIL protein (Fig. 4GoA, lanes 1 and 3); immunostaining with monoclonal anti-TRAIL Ab revealed that 52.9% of the PMA-treated Jurkat cells express TRAIL on their surface (Fig. 4GoB2). Hyperstimulation with a combination of either PMA/Con A, PMA/PHA, or PMA/ionomycin resulted in a striking increase in TRAIL protein expression with 75–80% of the cells expressing surface TRAIL (Fig. 4Go, B, 3–5, and A, lanes 9–13). In contrast with previous results (23), Con A, PHA, and ionomycin were unable to up-regulate TRAIL protein expression in the absence of PMA (Fig. 4GoA, lanes 4–8). Although TNF-{alpha} stimulation resulted in a 15-fold increase of TRAIL mRNA, a moderate 2-fold increase of TRAIL protein was observed (Fig. 4Go, A, lane 2, and B1).



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  		FIGURE 4. Expression of TRAIL protein on Jurkat T cells. A, The rtTA-Neo Jurkat T cells were induced for 48 h with either TNF-{alpha} (20 ng/ml), PMA (100 ng/ml), Con A (10 µg/ml), PHA (1 µg/ml), ionomycin (at 200, 500, and 1000 pmol/ml), or a combination of PMA/Con A, PMA/PHA, and PMA/ionomycin (same concentrations as above). Whole-cell extracts were subjected to immunobloting using the Immunex TRAIL-specific Ab. B, The rtTA-Neo cells were incubated as described above with either TNF-{alpha} (20 ng/ml), PMA (100 ng/ml), or a combination of PMA (100 ng/ml) with Con A (10 µg/ml), PHA (1 µg/ml), or ionomycin (200 pmol/ml). Expression of surface TRAIL was analyzed by flow cytometry using the Immunex TRAIL Ab.

 
Time-course experiments in which Jurkat cells were stimulated for various amounts of time with either PMA or PMA/Con A displayed detectable levels of TRAIL mRNA and protein in cell extracts within 2 h after PMA or PMA/Con A stimulation (Fig. 5Go, A and B, lanes 8 and 14). Levels of TRAIL mRNA and protein peaked 8–14 h after stimulation (Fig. 5Go, A and B, lanes 9–13 and 15–19) and remained detectable throughout the 48-h time course. Flow cytometric analysis of Jurkat T cells stimulated for 4 h with PMA and PMA/Con A confirmed these kinetics and revealed a 25- to 35-fold increase in surface TRAIL expression (Fig. 5GoC).



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  		FIGURE 5. Kinetics of TRAIL expression. A, The rtTA-Neo Jurkat T cells were induced for various amounts of time (0–48 h) with Con A (10 µg/ml), PMA (100 ng/ml), and PMA/Con A (at 100 ng/ml and 10 µg/ml, respectively). Total RNA was extracted and then subjected to RNase protection assay using the hAPO3c probe set. L32- and GAPDH-protected probes were used for normalization. B, The rtTA-Neo cells were treated as described above, and expression of TRAIL protein was analyzed by immunoblotting using the Immunex TRAIL Ab. C, The rtTA-Neo cells were induced with PMA (100 ng/ml) and PMA/Con A (100 ng/ml and 10 µg/ml, respectively) for 0, 4, 8, 12, and 24 h and were then subjected to flow cytometric analysis using the Immunex TRAIL Ab.

 
TRAIL expression in T lymphocytes is dependent on NF-{kappa}B

To determine whether PMA- and PMA/Con A-induced stimulation of TRAIL is mediated by NF-{kappa}B, rtTA-Neo and rtTA-I{kappa}B{alpha}(2N{Delta}4) cells were pretreated with Dox for varying amounts of time (0–24 h) and were subsequently stimulated with PMA or PMA/Con A for 8 h. RNA and protein were then extracted from these cells and subjected to RNase protection assay and immunoblot analysis, respectively. As expected, both PMA and PMA/Con A stimulation resulted in a marked increase in both TRAIL mRNA and protein (Fig. 6Go, A and B, lanes 3–6, 9, and 11). After induction of TD-I{kappa}B{alpha} for 24 h, a complete disappearance of TRAIL mRNA as well as TRAIL protein was observed (Fig. 6Go, A and B, lanes 10 and 12). Moreover, PMA- and PMA/Con A-induced expression of surface TRAIL was reduced by 5- to 10-fold in rtTA-I{kappa}B{alpha}(2N{Delta}4) cells upon treatment with Dox (Fig. 6GoC, compare 2 and 4, 5 and 7). Both PMA- and PMA/Con A-induced expression of surface TRAIL were weaker in rtTA-I{kappa}B{alpha}(2N{Delta}4) than in rtTA-Neo cells, which may be due to the leakiness of TD-I{kappa}B{alpha} expression (44). Similarly, PMA/ionomycin-induced activation of TRAIL mRNA and surface protein was also dramatically reduced following expression of TD-I{kappa}B{alpha} expression in rtTA-I{kappa}B{alpha}(2N{Delta}4) cells (data not shown).



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  		FIGURE 6. TRAIL expression in Jurkat T cells is dependent on NF-{kappa}B. The rtTA-Neo and rtTA-I{kappa}B{alpha}(2N{Delta}4) Jurkat cells were incubated with Dox for different intervals (0–24 h) and subsequently treated with PMA (100 ng/ml) or PMA/Con A (100 ng/ml and 10 µg/ml, respectively) for 4 h. A, Total RNA was extracted and subjected to RNase protection assay using the hAPO3c probe set. L32- and GAPDH-protected probes were used for normalization. B, Whole-cell extracts were analyzed for TRAIL expression by immunoblotting with the Immunex TRAIL Ab. C, The rtTA-I{kappa}B{alpha}(2N{Delta}4) Jurkat cells were incubated with Dox for 0, 12, and 24 h and were subsequently treated with PMA or PMA/Con A as described above. Expression of surface TRAIL was analyzed by flow cytometry using the Immunex TRAIL-specific Ab.

 
To evaluate the physiological significance of these results, primary CD3+ T cells obtained from the blood of healthy donors were stimulated with a variety of T cell activators. Anti-CD3/anti-CD28, PMA/Con A, and PMA/PHA stimulation of primary T cells resulted in an ~2- to 3-fold increase in TRAIL mRNA, as opposed to unstimulated cells. PMA/ionomycin stimulation resulted in a weaker activation, although we were able to observe a 1.5-fold increase in TRAIL mRNA expression (Fig. 7GoA). Immunofluorescence analysis of surface TRAIL revealed similar expression patterns, with 35–45% of cells expressing TRAIL following PMA/Con A and PMA/PHA stimulation and 12% of cells expressing TRAIL following PMA/ionomycin stimulation (Fig. 7GoA).



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  		FIGURE 7. TRAIL expression in primary T lymphocytes is dependent on NF-{kappa}B. A, Primary T lymphocytes were stimulated with a combination of anti-CD3/anti-CD28 (10 ng/ml and 5 µg/ml, respectively), PMA/Con A (100 ng/ml and 10 µg/ml, respectively), PMA/PHA (100 ng/ml and 1 µg/ml, respectively), or PMA/ionomycin (Ion) (100 ng/ml and 1000 pmol/ml, respectively) for 48 h (left panel). PMA/Con A-induced activation of NF-{kappa}B (100 ng/ml and 10 µg/ml, respectively, for 48 h) was blocked by the addition of NaSal (20 mM) and Bay 11-7082 (5 µM) at the 24-h time point of the stimulation or MG132 (10 µM) at the 44-h time point of the stimulation (right panel). Total RNA was extracted and subjected to RNase protection assay using the hAPO3c probe set. L32-protected probes were used for normalization. B, Primary T lymphocytes were stimulated with PMA/ionomycin (Ion), PMA/Con A, and PMA/PHA as described above. PMA/Con A-induced activation of NF-{kappa}B (100 ng/ml and 10 µg/ml, respectively, for 48 h) was blocked by the treatment of stimulated cells with NaSal (20 mM) for 24 or 48 h. Expression of surface TRAIL was analyzed by flow cytometry using the Immunex TRAIL-specific Ab.

 
To assess the importance of NF-{kappa}B during activation-induced expression of TRAIL, primary T lymphocytes were stimulated with a combination of PMA (100 ng/ml) and Con A (10 µg/ml) for 48 h in the absence or presence of three pharmacological inhibitors of NF-{kappa}B: NaSal and Bay 11-7082, which are known inhibitors of I{kappa}B phosphorylation, and MG132, a potent proteasome inhibitor (47, 48, 49). PMA/Con A stimulation resulted in an ~2-fold increase in TRAIL mRNA, as compared with resting cells. Addition of NaSal (20 mM) or Bay 11-7082 (5 µM) at 24 h after PMA/Con A stimulation resulted in a complete inhibition of the induced TRAIL mRNA expression (Fig. 7GoA). Incubation of PMA/Con A-stimulated primary T lymphocytes for 4 h with the proteasome inhibitor MG132 (10 µM) also resulted in a complete abrogation of TRAIL mRNA up-regulation (Fig. 7GoA). Moreover, treatment of PMA/Con A-stimulated T cells with NaSal for 24 and 48 h resulted in a drop from 40% of cells expressing TRAIL to 26 and 14%, respectively (Fig. 7GoB). Taken together, these results demonstrate an important role for NF-{kappa}B in the activation of TRAIL expression in both Jurkat T cell lines and primary T lymphocytes.

TRAIL promoter analysis

Analysis of TRAIL expression at the mRNA and protein levels provided strong evidence for an NF-{kappa}B-dependent mechanism regulating TRAIL gene expression. Computer sequence analysis revealed the presence of two potential NF-{kappa}B binding sites, {kappa}B1 and {kappa}B2, in the proximal 538 bp of the TRAIL promoter region, as well as a more distant {kappa}B3 site that spans from -1317 to -1327 (Fig. 8GoA). PMA-induced NF-{kappa}B binding was studied by EMSA using all three sites and extracts from rtTA-I{kappa}B{alpha}(2N{Delta}4) and rtTA-Neo cells. PMA stimulation for 16 h resulted in a strong induction of NF-{kappa}B binding on the {kappa}B1 site (Fig. 8GoB, lanes 2–4 and 7), whereas induced binding on the {kappa}B2 and {kappa}B3 sites was not detected (data not shown). In contrast, treatment of rtTA-I{kappa}B{alpha}(2N{Delta}4) cells with Dox completely abrogated PMA-induced NF-{kappa}B binding (Fig. 8GoB, compare lanes 7 and 8), whereas Dox treatment did not affect binding in rtTA-Neo cells (Fig. 8GoB, compare lanes 3 and 4). To identify the subunit composition of the NF-{kappa}B complexes, supershift analysis was performed using anti-p65, anti-p50, anti-c-Rel, and anti-RelB Abs. Induced complexes were shifted with p50 and c-Rel Abs (Fig. 8GoB, lanes 18 and 20), but not by p65 or RelB Abs (lanes 16 and 21), indicating the presence of p50 and c-Rel heterodimers in PMA-induced complexes. In addition, p50 dimers were able to bind the probe in the absence of any stimulation (Fig. 8GoB, lanes 17 and 18). A mutated {kappa}B1 site in which the c-Rel consensus site was altered failed to bind NF-{kappa}B following PMA stimulation (Fig. 8GoB, lanes 9 and 10).



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  		FIGURE 8. c-Rel binds the TRAIL promoter in vitro. A, Schematic representation of the TRAIL promoter region and sequence of the proximal promoter region. B, Nuclear extracts from rtTA-Neo (lanes 1–4) and rtTA-I{kappa}B{alpha}(2N{Delta}4) Jurkat cells (lanes 5–8) treated or untreated with PMA (100 ng/ml) for 16 h in the absence or presence of Dox pretreatment (1 µg/ml, 48 h) were subjected to an EMSA using the TRAIL {kappa}B1 probe. Binding of uninduced and PMA-induced complexes to a mutated version of the TRAIL {kappa}B1 probe was also tested (lanes 9 and 10). Nuclear extracts from rtTA-Neo cells either untreated or treated with PMA (100 ng/ml) for 16 h were subjected to supershift analysis using the Santa Cruz p65, p50, c-Rel, and RelB Abs (1 µg/reaction) (compare lanes 15–22 with control lanes 11–12). Lanes 23–24, The NF-{kappa}B-DNA complex formation was competed using a 125-fold excess of unlabeled TRAIL {kappa}B1 probe. Lanes 13–14, Complexes treated with control rabbit serum (1 µg/reaction).

 
To verify the role of the NF-{kappa}B sites in TRAIL regulation, the 5' 1578-bp region flanking the TRAIL gene (GenBank accession no. AF178756) was cloned from genomic DNA into CAT and luciferase reporter plasmids. This fragment contains the transcription start site of the TRAIL mRNA (50); in addition, two shorter fragments containing the sequences up to -1002 and -483 bp flanking the TRAIL gene were also cloned. Preliminary results showed that the -483 TRAIL promoter fragment was sufficient for PMA-induced transactivation of a luciferase reporter construct (data not shown). CAT reporter activity was measured following PMA stimulation in the absence or presence of TD-I{kappa}B{alpha} expression. As shown in Fig. 9GoA, PMA stimulation resulted in an ~3.5 fold induction of reporter gene activity, whereas Dox treatment of cells resulted in a complete loss of PMA-dependent up-regulation of reporter activity. It should be noted that Dox treatment had no effect on the basal levels of promoter activity, and only the induced activity of the promoter was affected. To eliminate the possibility that NF-{kappa}B may act through an indirect mechanism to transactivate TRAIL, de novo protein synthesis was blocked using cycloheximide, and TRAIL levels were measured in uninduced and PMA-induced cells. Treatment of cells with both cycloheximide and PMA resulted in an ~4-fold increase in TRAIL mRNA and protein levels as compared with cells treated with cycloheximide alone, indicating that the observed up-regulation of TRAIL was, at least in part, directly mediated by NF-{kappa}B (data not shown).



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  		FIGURE 9. TRAIL promoter analysis. A, The rtTA-I{kappa}B{alpha}(2N{Delta}4) Jurkat cells were transfected with CAT reporter constructs containing the -483 TRAIL promoter region or with the empty pCAT basic vector (10 µg/107 cells). Cells were then incubated with Dox for different amounts of time (0 or 24 h) followed by induction with PMA (100 ng/ml for 0 or 16 h). CAT activity was plotted as fold induction compared with the empty pCAT basic vector activity. Mock, Mock-transfected cells. B, Luciferase constructs containing the wild-type and mutated -483 TRAIL promoter region (1 µg/106 cells) were cotransfected with pRL-tk (100 ng/106 cells) and either a control or NIK expression vector (2.9 µg/106 cells) in Jurkat T cells. Luciferase activity was measured 48 h following transfection and was plotted as fold induction of NIK-transfected samples compared with control (empty) expression vector-transfected samples for each TRAIL promoter construct.

 
To assess the importance of the NF-{kappa}B sites within the proximal TRAIL promoter, luciferase constructs containing either a mutated {kappa}B1 (-483 TRAIL {kappa}B1 mutant) or {kappa}B2 (-483 TRAIL {kappa}B2 mutant) site or a combined mutation of both {kappa}B1 and {kappa}B2 sites (-483 TRAIL {kappa}B1/{kappa}B2 mutant) were generated. Although PMA stimulated the wild-type promoter ~4-fold, the stimulation remained relatively low, due in part to the cytotoxic effect of PMA on Jurkat cells. To avoid this difficulty, cells were activated by overexpressing NIK, a well-characterized activator of the NF-{kappa}B pathway (51). Cotransfection of a NIK-expressing plasmid in Jurkat T cells resulted in 5-fold induction of the -483 TRAIL promoter fragment, as compared with the cells transfected with the empty control plasmid (Fig. 9GoB). In contrast, expression of NIK did not activate a promoter containing a mutation in the {kappa}B1site. Surprisingly, a {kappa}B2-mutated promoter also resulted in a complete loss of NIK-induced activity, despite the inability to detect any induced NF-{kappa}B binding to this site. Given the close proximity of the {kappa}B1 and {kappa}B2 sites, this result may reflect the requirement for cooperative binding to these two sites, with binding to the {kappa}B1 site increasing the probability of binding to the {kappa}B2 site. Significantly, the double mutant promoter, in which both the {kappa}B1 and the {kappa}B2 sites were mutated, showed no increase in activity following NIK overexpression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The cytotoxic effect of various immune cells, including CD4+ T cells, NK cells, macrophages, and dendritic cells, is at least partially dependent on the expression of TRAIL, as well as on Fas ligand (FasL) expression (18, 52, 53, 54). Although regulatory mechanisms leading to the induction of FasL have been delineated, the molecular components involved in TRAIL expression following T cell activation remain poorly understood. In this present study, we demonstrate that NF-{kappa}B directly up-regulates TRAIL, another member of the TNF family of cellular ligands, which includes FasL and TNF-{alpha}. Cell surface expression of TRAIL was increased in Jurkat T cells following treatment with a variety of stimuli such as TNF-{alpha}, PMA, PMA/Con A, PMA/PHA, or PMA/ionomycin. In Jurkat T cells stably expressing the TD-I{kappa}B{alpha}, both TRAIL mRNA and protein expression were dramatically reduced; in parallel, we also demonstrated that pharmacological inhibitors of NF-{kappa}B were able to abrogate T cell activation-induced expression of TRAIL in primary T lymphocytes. NF-{kappa}B-dependent expression of TRAIL was linked to the presence of a c-Rel-p50 NF-{kappa}B binding site located between -256 and -265 within the TRAIL promoter; a second putative adjacent site was also identified, but this site failed to bind an inducible NF-{kappa}B complex. Interestingly, mutation of these two sites resulted in a complete loss of inducibility of the TRAIL promoter.

A number of important genes involved in immunoregulation and cytokine and chemokine gene expression are under the control of NF-{kappa}B. Moreover, NF-{kappa}B has been shown to play an important role in regulating antiapoptotic and proapoptotic events, depending on the physiological circumstances (55). For example, NF-{kappa}B is actively involved in activation-induced cell death (AICD) of mature T cells by up-regulating the expression of FasL. Indeed, in the present studies, activation of NF-{kappa}B following TCR engagement resulted in the increased expression of both TRAIL and FasL (Fig. 7Go). Furthermore, the promoter of human FasL contains {kappa}B sites and can be up-regulated by AICD in T cells (56, 57). Similarly, modulation of expression of FasL on T cells can influence T cell-mediated apoptosis of autoreactive B cells (58). Thus, by regulating apoptosis-inducing ligands, NF-{kappa}B may play an important role in preventing autoimmunity. In addition, activated T cells are also important effectors of immunological defense against tumors, and one potential mechanism to eliminate malignant tumors may be to induce apoptosis via TRAIL, FasL, or other TNF family ligands (59).

Like other members of the TNF ligand family, TRAIL is able to induce apoptosis in a variety of cell lines (2). Interestingly, TRAIL preferentially induces apoptosis in various tumor cell lines but not in normal cells (60), suggesting that TRAIL may have therapeutic potential. Members of the TNF family are involved in the modulation of host defense mechanisms including T cell costimulation, induction of B cell proliferation, macrophage activation, as well as elimination of unwanted immune cells by apoptosis (61). Recent studies revealed that the cytotoxic effect of a variety of immune cells including CD4+ T cells, NK cells, macrophages, and dendritic cells is at least partly dependent on TRAIL expression, suggesting a potential role for TRAIL as a tumor suppressor (18, 52, 53, 54). It has also been suggested that TRAIL may play an important role in virus-induced apoptosis; in this regard, TRAIL may be responsible for the AICD of T cells during HIV infection (62, 63). Related studies also demonstrate that Reovirus-, measles virus-, and human CMV-infected cells are rendered cytotoxic via the TRAIL pathway (64, 65, 66) and indicate that virus-infected cells express enhanced levels of TRAIL, which is responsible, at least in part, for virus-induced apoptosis (62, 63, 64, 65, 66). Given the results of the present study, we conclude that TRAIL expression may be enhanced as a result of viral-dependent activation of NF-{kappa}B.

The involvement of other transcriptional activators, such as the IFN regulatory factors (IRFs) and/or NF-AT proteins, would provide an additional level of complexity in the regulation of TRAIL surface expression. Sequence analysis of the TRAIL promoter also revealed several potential NF-AT and AP-1 sites; it is well established that the NF-AT sites contained within the TNF-{alpha} and FasL promoters are important in up-regulation following T cell activation (56, 67). The increased expression of surface TRAIL in the presence of a combined action of phorbol ester plus ionomycin stimulation, as opposed to phorbol ester alone, could argue for a combined NF-{kappa}B-NF-AT action. However, this could also be explained by the fact that the signaling requirements for c-Rel mimic those of NF-AT. Whereas p65 can be efficiently induced by phorbol ester alone, optimal induction of c-Rel requires additional stimulation, such as phorbol ester and ionomycin (68, 69). Previous experiments have also revealed that TRAIL can be up-regulated following IFN treatment (18, 19, 20), suggesting an additional level of regulation mediated by IRFs. A similar cooperative regulatory mechanism can be observed in the case of the FasL, which is regulated by NF-{kappa}B and IRF-1/IRF-2 (70). Thus, regulation of TRAIL in T cells may parallel in many respects that of FasL or other members of the TNF family and may include a requirement for NF-AT and IRFs following physiological or pharmacological activation. Nonetheless, the present study illustrates the absolute requirement for NF-{kappa}B in Ag receptor-induced expression of TRAIL in T lymphocytes.


   Acknowledgments
 
We thank members of the Molecular Oncology Group, Lady Davis Institute for helpful discussions.


   Footnotes
 
1 This research was supported by grants from the Canadian Vaccine Initiative the National Cancer Institute of Canada, and from the Canadian Vaccine Institute Network Centers of Excellence. T.M.B., H.K., and S.S. were supported by Fonds pour la Formation de Chercheurs et l’Aide à la Recerche Studentships, N.G. was supported by Association pour la Recherche sur le Cancer and Fonds de la Recherche Scientifique Médicale (Quebec) postdoctoral fellowships, and J.H. was supported by Canadian Institutes for Health Research Senior Scientist award. Back

2 Address correspondence and reprint requests to Dr. John Hiscott, Lady Davis Institute for Medical Research, McGill University, 3755 Cote Ste. Catherine, Montreal, Quebec, Canada H3T 1E2. E-mail address: jhisco{at}po-box.mcgill.ca Back

3 Abbreviations used in this paper: TRAIL, TNF-related apoptosis-inducing ligand; FADD, Fas-associated death domain; IKK, I{kappa}B kinase; NIK, NF-{kappa}B-inducing kinase; DcR, decoy receptor; TD-I{kappa}B{alpha}, transdominant repressor of I{kappa}B{alpha}; NaSal, sodium salicylate; Dox, doxycycline; NP-40, Nonidet P-40; CAT, chloramphenicol acetyltransferase; rtTA, reverse tetracycline transactivator protein; FasL, Fas ligand; AICD, activation-induced cell death; IRF, IFN regulatory factor. Back

Received for publication April 6, 2001. Accepted for publication July 10, 2001.


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